Pharmacokinetic Study of Coadministration with Cefuroxime Sodium for Injection Influencing ReDuNing Injection-Derived Seven Phytochemicals and Nine Metabolites in Rats

According to the sixth edition of China's “New Coronavirus Diagnosis and Treatment Plan (NCDTP),” ReDuNing injection (RDN) was firstly introduced to treat severe and critical COVID-19, whereas its combination with broad-spectrum antibiotics was suggested to take with extreme caution and full reasons. Therefore, we aim to describe the pharmacokinetics of seven active phytochemicals and semiquantification of nine relevant metabolites in ReDuNing injection (RDN) after combining with cefuroxime sodium (CNa) for injection in rat plasma. Male Sprague–Dawley rats were randomly assigned to six groups, and they were intravenously administered, respectively, with different prescriptions of RDN (2 mL/kg) and CNa (225 mg/kg). At different time points (0.03, 0.08, 0.17, 0.24, 0.33, 0.50, 0.67, 1, and 6 h) after administration, the drug concentrations of iridoids glycosides, organic acids, and metabolites in rat plasma were determined using ultrahigh-pressure liquid chromatography coupled with linear ion rap-orbitrap tandem mass spectrometry (UHPLC–LTQ–Orbitrap–MS), and main pharmacokinetic parameters were estimated by noncompartment model. The results showed that there were differences in pharmacokinetic parameters, AUC(0-t), T1/2, Cmax, CL of iridoids glycosides, and organic acids, after the intravenous administration of the different combinations of RDN and CNa. Moreover, different combinations of the injections also resulted in different curves of relative changes of each metabolite. The obtained results suggested that RDN and CNa existed pharmacokinetic drug–herb interactions in rats. The findings not only lay the foundation for evaluating the safety of RDN injection combined with CNa but also make contributions to clinically applying RDN injection combined with CNa, which works potentially against severe forms of COVID-19.


Introduction
Since December 2019, the outbreak of COVID-19 has caused over 196,000,000 confirmed cases with more than 4,100,000 deaths worldwide as of July 29, 2021, according to statistical data from the WHO and Hopkins University website (https://nxw.so/5JfTZ). To fight against its ravages, more than 280 candidate vaccines are currently in development, 23 of which are already in phase 3 trials through different platforms [1]. However, COVID-19 also evolved into an "improved" version, i.e., the Delta variant identified in October 2020 in India [2], with ∼60% more transmissible properties than the already highly infectious Alpha variant [3]. As a result, a single dose of either Pfizer or AstraZeneca vaccines barely induced neutralizing antibodies against the Delta variant in individuals who were not previously infected with SARS-CoV-2 [4]. Some scholars think that it is merely a matter of time before the Delta becomes dominant and takes over, however, the hope is to slow its expansion through vaccination [3]. In addition, severe systemic events (vomiting, diarrhea, fatigue, headache, chills, muscle pain, joint pain, etc.) were reported when using the above two vaccine candidates [5]. Although a second dose of the AstraZeneca vaccine boosted protection against Delta to 60% [3], it remains worrisome and uncertain what the vaccine efficacy against Delta will be for those more severe forms of disease [6].
Currently, several clinical reports have involved RDN against bronchitis and pneumonia in combination with cefuroxime sodium for injection (CNa, a semisynthetic cephalosporin with relatively broad-spectrum antimicrobial activity) [16][17][18][19], which effectively inhibits viruses or bacteria, removes inflammatory mediators, regulates patient immunity, and improves patient symptoms [20,21]. However, such a combination may result in severe medicinal accidents because of the in vivo drug-herb interaction causing changes in the blood concentration and metabolites [22][23][24][25][26], to which we should pay attention in the medication guide of RDN (https://nxw.so/5x4Pz).
To ensure safety and efficacy, qualitative and quantitative analyses of RDN-derived major components have been processed using near-infrared spectroscopy and UPLC methods [27,28]. In addition, there have been several reports on in vivo pharmacokinetic studies of some iridoid glycosides and organic acids, such as geniposide, neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid, in RDN-administered humans or rats [29][30][31][32][33][34]. Although most research has made efforts to develop an effective method to study the in vivo profile of prototypes in RDN, knowledge on not only the pharmacokinetics of RDN combined with other drugs but also the change trends of relevant metabolites are still limited. Given the absence of in vivo studies reporting RDN injection combined with CNa, we aimed to evaluate the changes in the pharmacokinetic profiles of prototypes and major metabolites of RDN with CNa after coadministration. is study mainly focused on the interaction of the two injections with regards to pharmacokinetics using UHPLC-LTQ-Orbitrap-MS, laying the foundation of drug combination of RDN injection with CNa and making a contribution to preparing a potential therapeutic for severe forms of COVID-19.

Materials and Methods
2.1. Chemicals and Reagents. RDN injection was provided by the Jiangsu Kanion Pharmaceutical Co., Ltd, and CNa was obtained from the Shenzhen Lijian Pharmaceutical Co., Ltd. Reference standards (geniposide, shanzhiside, genipin-1β-gentiobioside, secoxyloganin, neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid) were provided by the Shanghai Rongqi Pharmaceutical Technology Co., Ltd. (Shanghai, China). Clarithromycin (IS, 98% purity, 1529013) was purchased from the Shanghai Aladdin Bio-Chem Technology Co., Ltd. Cefuroxime was obtained from the National Institutes for Food and Drug Control. e relevant chemical structures are shown in Figure 1. e mass spectrum of seven ingredients of RDN and cefuroxime are shown in Figure 2. Acetonitrile, methanol, and formic acid were of HPLC grade from Merck (Darmstadt, Germany). Deionized water was purified by a Milli-Q system (Millipore, Billerica, MA, USA). e other chemical reagents were of analytical grade.
A total of 7 components in RDN injection were determined.
e stock solutions of all analytes were combined and further diluted with methanol water (50 : 50, v/v) using 2, 5, 2, 5, 2 and 5-fold serial gradient. 200 μL of the combined working solution was added to 50 μL plasma and 20 μL IS (1 μg·mL −1 ) to obtain the calibration standards at 62,

Sample Preparation.
A 50 μL aliquot of plasma was mixed with 200 μL of methanol and 20 μL of IS (2) [37]. e solution was vortexed for 2 min and centrifuged at 13,000 rpm for 10 min. e clarified supernatant was transferred to a new polypropylene tube and evaporated to dryness under nitrogen at 30°C. e residue was reconstituted in 50 μL acetonitrile-water (5 : 95 v/v), vortex-mixed, and centrifuged again under the above-mentioned conditions [38]. 2 μL of this solution was injected into the UPLC-MS/MS for analysis [39].

Method Validation of Prototypes.
e proposed quantitative method was validated by determining the selectivity, linearity, precision, accuracy, extraction recovery, matrix effect, and stability according to the guidance of the Food and Drug Administration (FDA) for the validation of bioanalytical methods [40].

Selectivity.
e selectivity of the method was evaluated by comparing the chromatograms of blank plasma samples with those of corresponding standard samples spiked with analytes, IS, and samples.

Linearity and Lower Limit of Quantification.
e linear calibration curves of seven analytes with cefuroxime were determined by plotting the peak area ratio (y) of analytes to IS versus the analyte concentration (x) by leastsquares linear regression using 1/x 2 as the weighing factor. e calibration curves had to have a correlation coefficient (R) of 0.99 or better. e limit of detection (LLOD) and quantification (LLOQ) were determined at signal-to-noise ratios of 3 and 10 by analyzing the standard plasma samples.

Precision and Accuracy.
e precision and accuracy depended on analyzing QC samples at three different concentration levels (low, medium, and high) in six replicates. e RSD was used for reporting precision. e accuracy was established by comparing the measured concentration with its true value. Accuracy and precision were assessed by the relative error (RE) and relative standard deviation (RSD), respectively. Precision should not exceed 15%, and the accuracy should be within ±15% for the QC samples.

Extraction Recovery and Matrix Effect.
e extraction recovery of the seven prototypes with cefuroxime at three QC levels was evaluated by comparing the peak area of each analyte extracted from plasma QC samples with those latter extracted from the blank matrix (n � 6). e matrix effect was determined as the peak area of the analytes dissolved in the blank matrix versus that dissolved with methanol solution [41].

Stability.
e stability of seven analytes with cefuroxime in plasma was evaluated by keeping the low-, medium-, and high-QC samples at 25°C for 4 h (short-term stability), storing the samples at −20°C for 21 days (longterm stability), and undergoing three freeze/thaw cycles. e autosampler stability was evaluated by analyzing QC samples at 4°C for 24 h.

Animal Treatment.
Experimental animals were randomly divided into eight groups (six rats per group), which are listed in Table 2. e doses of RDN and CNa based on clinical practice were 2 mL/kg and 225 mg/kg, respectively. Groups 1/2/3 received RDN/CNa/coadministration (RDN and CNa) alone merely for one day, while Groups 4/7/8 received RDN/CNa/coadministration (RDN and CNa) for 5 consecutive days. Group 5 received CNa on the first 4 days and then RDN on the 5 th day. Group 6 received 4-day coadministration of RDN and CNa and RDN alone on the last day. All of the above drugs were administered in the form of intravenous injection through the caudal vein, and there was no interval between drug-herb administration [42]. Serial blood samples (300 μL) were obtained on the last day at 0 (predose), 0.03, 0.08, 0.17, 0.24, 0.33, 0.50, 0.67, 1, and 6 h after intravenous injection. After centrifugation at 4500 rpm for 10 min, plasma was collected and frozen at −20°C until analysis.

Statistical Analysis.
Pharmacokinetic parameters were determined using Drug and Statistic (DAS) software (version 2.0, Chinese Pharmacological Society). Parameters, including the terminal elimination half-life (T 1/2 ), maximum plasma concentration (C max ), area under the plasma concentration-time curve (AUC 0−t ), and plasma clearance (CL), were determined. Data were presented as the mean-± standard deviation (SD). Student's t-test was used for the comparisons of two groups, except when the variances of the compared groups were not homogeneous, in which case the Mann-Whitney U test was employed.

Method Validation for the Semiquantification of Metabolites
(1) Linear Range. A semiquantitative method for nine metabolites was built because of the lack of reference standards. Samples from every time point were mixed together (150 μL per sample) as the mother solution [44]. e mother solutions of 5 μL, 10 μL, 20 μL, 40 μL, 80 μL, 120 μL, and 200 μL were added to 1.5 mL centrifuge tubes, and then, 200 μL of each tube was added to 200 μL of blank plasma. Every tube was supplemented with 80 μL of IS solution (1 μg/mL) and 400 μL of methanol. en, 600 μL of supernatant was collected after centrifugation at 13,000 rpm for 10 min and concentrated by centrifugation. Next, the residue was dissolved by 50 μL of 5% acetonitrile. ese seven samples aimed to establish the linear range of each metabolite curve. Samples with 15 μL, 40 μL, and 160 μL of mother solution were used as QC samples.
(2) Precision and Stability. e precision of metabolites was evaluated by measuring QC samples at low, medium, and high concentrations, expressed as the RSD, which should not exceed 15%. e stability of metabolites was estimated as described in "Section 2.5.5."

Curves of Relative Changes for the Metabolites.
e curves of relative changes of these metabolites during 6 h after intravenous injection were constructed by plotting the peak area ratios of metabolites to the IS (X-axis is time and Y-axis is the peak area ratios of metabolites to IS), respectively.

Method Validation of Prototypes
e protein precipitation methodology through mass spectrometry detection presented good selectivity for the analytes. Typical chromatograms obtained from a blank, a spiked plasma sample with the seven analytes, cefuroxime, and the IS after an intravenous dose are shown in Figure 3. Significant interference from endogenous components was hardly found to affect the detection of the analytes and IS in all samples.

Linearity and Lower Limit of Quantification.
Regression equations, linear ranges, correlation coefficients and LLOQs for the seven analytes with cefuroxime are shown in Table 3. e assay exhibited good linearity for all constituents with correlation coefficients in the range from 0.9990 to 0.9996.

Extraction Recoveries and Matrix Effects.
e results of the matrix effect and extraction recovery are summarized in Table 4. e recoveries of the analytes extracted from the plasma at the three QC concentration levels were 90. 36

Precision and Accuracy.
e precision and accuracy were confirmed by assaying the QC samples at three concentration levels, as listed in Table 4. Both the intraday and interday precision of the QC samples in the plasma were below 11.49% at each level, and the accuracy of chemical ingredients ranged from −7.73% to 9.22%. All of these values were within the acceptable range, and the method was judged to be suitably accurate and precise.

Stabilities.
e results of the stability study, as shown in Table 5, suggested that the analytes in the plasma maintained good short-term stability, long-term stability, freeze-thaw stability, and autosampler stability.

Pharmacokinetic Study of Prototype Compounds.
e above validated UHPLC-LTQ-Orbitrap-MS method was applied successfully to the pharmacokinetic study in the rat plasma for the respective groups. e plasma concentrationtime profiles of seven analytes with cefuroxime in single combination and multiple combination are illustrated in    e pharmacokinetic parameters are shown in Table 6. Representative extract ion chromatograms and MS data of metabolites are shown in Figure 6 and Table 7, respectively. e structures of metabolites are shown in Figure 7.

Semiquantitative Method for
Metabolites. e proposed semiquantitative method was validated by linear range, precision, and stability tests, and the results are shown  Tables 8 and 9.
e method satisfied the demands of semiquantification for the metabolites.

Curves of Relative Changes for Metabolites.
e curves of relative changes of these metabolites during 6 h are displayed in Figures 8 and 9.

LC-MS Optimization.
To develop a sensitive method, all analytes were full-scanned by the positive and negative modes. It was found that the analytes could be ionized under both modes. e mass spectrometric parameters, such as the source voltage and heater temperature, capillary temperature, the flow rate of sheath gas, and the flow rate of auxiliary gas, were optimized to obtain the highest signal for the precursor ions and product ions mentioned above. (Group 1 and Group 3). Iridoid glycosides in plasma showed a converse trend to organic acids after combination with CNa in pharmacokinetics. e one-time coadministration of RDN and CNa led to lower plasma concentrations of iridoid glycosides, with reduced AUC (0-t) and C max values, respectively, especially geniposide. Compared with the administration of RDN alone (Group 1), the AUC (0-t) and C max values for geniposide in Group 3 were reduced by 14.5% and 26.8%, respectively. T 1/2 values were shortened from 0.50 to 0.27 h, and the three parameters were considered to be significant (p < 0.05) after the analysis of  variance. e differences in two parameters, AUC (0-t) and C max , of organic acids (neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid) in plasma were appreciably increased by 1.2-2.1 times after injecting RDN combined with CNa, and CL values also decreased significantly (p < 0.05). Meanwhile, the T 1/2 was also decreased, however, there were no remarkable disparities in the values. CNa is not metabolized and is principally excreted unchanged in urine in terms of both glomerular filtration and tubular secretion [45,46]. Such cephalosporin tubular reabsorption across brush-border membranes is mediated by pH-dependent peptide transporters PEPT1, PEPT2, and OAT [46][47][48]. As a good substrate of the above relevant transporters, CNa at higher concentrations can competitively block the uptake of organic acids with similar renal elimination [47][48][49], resulting in and adjunctively enhancing the blood level of organic acids [50].

Pharmacokinetics of RDN by Comparing the 5-Day Separate Administration of RDN with the 5-Day Coadministration of RDN and CNa (Group 4 and Group 8).
e plasma concentrations of iridoid glycosides and organic acids in rat serum were measured on the 5 th day and were lower in the presence of CNa (Group 8) than in its absence (Group 4). In the case of AUC (0-t) , the reductions of shanzhiside, genipin-1-β-gentiobioside, and secoxyloganin in rat plasma were up to 51.3% compared with the 5-day administration of RDN (p < 0.05), while geniposide had no significant changes. e trend of the AUC (0-t) parameter in organic acids was opposite to that observed when the two drugs were coadministered once (Group 1 and Group 3). Compared with the 5-day RDN administration, the AUC (0-t) values of the three chlorogenic acid isomers were reduced by nearly 30% under 5-day coadministration. Moreover, the C max of neochlorogenic acid in Group 8 declined by 30 Figure 7: e structures of metabolites in rat plasma. more than two times higher than those of chlorogenic acid and cryptochlorogenic acid. Similar to the first part pharmacokinetics, there were also no significant changes in the T 1/2 values of these isomers.

Pharmacokinetics of RDN by Comparing a 4-Day Administration of RDN and 1-Day CNa with a 5-Day Administration of RDN (Group 4 and Group 5).
In contrast to Group 4, the 5-day administration cycle of Group 5 consisted of 4-day RDN and 1-day CNa to fully simulate the combined administration in the clinic. e AUC (0-t) , C max , and CL of iridoid glycosides declined significantly (p < 0.05). e T 1/2 values of dosed geniposide, shanzhiside, and genipin-1-β-gentiobioside were 0.19-0.23 h in Group 5, which were significantly lower than those of Group 4 (T 1/ 2 > 0.36). e pharmacokinetic parameters, including AUC (0-t) and C max, in Group 5, displayed a 3-fold decrease for neochlorogenic acid, a 5-fold decrease for chlorogenic acid, and a 5-fold decrease for cryptochlorogenic acid. e rates of the decline were more than twice those of iridoid glycosides, indicating that CNa mostly affected the organic acids.

Pharmacokinetics of RDN by Comparing a 4-Day Coadministration and 1-Day RDN with 5-Day Separate Administration (Group 4 and Group 6).
e rats in Group 6 were treated with RDN combined with CNa on the first four days and administered RDN on the 5 th day. e AUC (0-t) values of geniposide, shanzhiside, genipin-β-1gentiobioside, and secoxyloganin in Group 6 decreased by nearly half those of Group 4. Except for secoxyloganin, the T 1/2 and C max of the other three iridoid glycosides significantly (p < 0.05) decreased. Similar to the iridoid glycosides, the pharmacokinetic parameters showed decreases of 62.0-70.8% in the AUC (0-t) of organic acids and decreases of 50.7-62.8% in C max compared with Group 4. In this study, the obtained pharmacokinetic parameter data for iridoid glycosides and organic acids in different groups showed differences. e above results suggest that the systemic exposure (AUC (0-t) and C max ) of geniposide, shanzhiside, genipin-1β-gentiobioside, and secoxyloganin were all weakened, and the elimination increased under the different coadministration conditions. CNa could be similar to its series cefetamet-inducing CYP3A4 enzyme activity [51], resulting in the fast metabolism of iridoid glycosides in rat plasma, however, further experiments are needed to prove this hypothesis. Meanwhile, CNa also affected the relative changes of the metabolites from geniposide and secoxyloganin.
e ring-opening, dehydration, and hydroxymethylene loss metabolites achieved lower C max values than others when coadministered with CNa one time. In Group 6 (RDN/CNa-4 + RDN-1), the peak time lag phenomenon was more obvious, which indicated that 4-day coadministration might inhibit these metabolic pathways in vivo.
e general trends of AUC (0-t) and C max , two important parameters of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in rat plasma, increased after the intravenous administration of RDN combined with CNa, and CL values decreased compared with RDN alone, indicating that single coadministration might improve the bioavailability of organic acids. As reported, CNa is not metabolized and is excreted unchanged by the renal anionic transport system, principally in urine by both glomerular filtration and tubular secretion [45,52]. Organic acids are negative ions and are mainly eliminated by the kidneys, which can cause competitive inhibition with cefuroxime from the blood to the kidney [53], which might be the reason for extending the exposure time after a single coadministration. e results of the groups administered for 5 days inferred that long-term drug interactions could increase the elimination of organic acids in rat plasma, indicating that CNa might shorten the potency of organic acids in vivo. It was reported that combining antibiotics for a long time can cause bacterial group maladjustment in vivo [54,55].

Influence of Coadministration with RDN on the Pharmacokinetic Profile of CNa.
Compared with Group 2, the concentration-time profiles of CNa showed faster elimination after the intravenous administration of rats in Group 3 (RDN/CNa-1). e pharmacokinetic parameters of CNa showed an 11.4-33.0% decrease in AUC (0-t) and a 9.9-72.5% increase in CL (all p < 0.05) as a consequence of a single combination with CNa. In addition, a 5-day coadministration with RDN (Group 8), also similar to the above single administration, decreased the AUC (0-t) and increased the CL of CNa compared with Group 7 but with no significant difference. Some published papers have reported that genipin enhances Mrp-2-mediated bile formation and organic anion transport [56], through which approximately 45% of the antibiotic cefuroxime is eliminated renally [57], while iridoid glycoside and geniposide can promote bile secretion [58,59], through which the cumulative percentage of cefuroxime reaches 12.83% [60].

Conclusion
A highly sensitive method using ultrahigh-pressure liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap-MS) has been developed and validated to characterize the RDN and CNa drug-herb pharmacokinetics and semiquantification of RDN metabolites in rat plasma. e results demonstrated that RDN combined with CNa could lead to lower systemic exposure of iridoid glycosides, while one coadministration could lead to the slower elimination of organic acids but faster elimination in 5-day different combinations of coadministration. e curves of the relative changes of nine metabolites of geniposide and secoxyloganin over 6 h were obtained. e pharmacokinetic results not only lay the foundation for evaluating the safety of RDN injection combined with CNa but also contribute to clinically applying RDN injection combined with CNa, which potentially protects against severe forms of COVID-19.

Data Availability
A majority of the data used in this research are included in the article. Other data can be made available upon request from the first author and corresponding author.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.